Quantifying the two populations of dual oriented P6 in nontypable Haemophilus influenzae

Abstract

Nontypable Haemophilus influenzae (NTHi) causes otitis media (ear infections) and other respiratory illnesses. Currently, NTHi's outer membrane protein P6 is a leading vaccine candidate for the pathogenic bacteria. Previous work in our lab has demonstrated that P6 is not a transmembrane protein. However, P6 is also known to interact with peptidoglycan inside the cell. In light of other studies performed in our lab (flow cytometry and confocal microscopy), which demonstrate P6's surface exposure, we conclude that P6 exists in two orientations in the outer membrane of NTHi. To quantify the two populations of inside‐facing and outside‐facing P6, we utilized a biotinylating reagent (NHS‐LC‐LC‐ biotin) to isolate surface exposed proteins from internally localized proteins. Standard protein detection and immunoblotting techniques were utilized to quantify the two populations of P6. We conclude that the predominant population of P6 is located on the inside of NTHi (>;75%). Further elucidation of the dual orientation of P6 and other lipoproteins is certainly important for both the fields of bacteriology and vaccine development. This study was funded by the Rochester Institute of Technology and NIH NIDCD RO1 08671 (to MEP).